Summary A common theme in bacterial evolution is how the acquisition of antibiotic resistance can rapidly change the epidemiologic landscape of major human pathogens. Carbapenem Resistant Klebsiella pneumoniae (CRKp), first documented in 1996, is now epidemic in New York City (NYC) hospitals and is reported globally. CRKp infections often result in poor therapeutic indices; thus curbing the incidence of CRKp infections is now a national priority. Currently, three overlapping CRKp epidemics with three different classes of carbapenemases are spreading in different continents. Accelerating these epidemics is the ability of carbapenemase genes, harbored on conjugative plasmids, to spread across the Enterobacteriaceae family. A critical, poorly understood aspect of the CRKp epidemic is the relative contribution of plasmid-mediated transfer and clonal dissemination to driving the regional and global epidemiology. Previously, we used whole genome sequencing (WGS) to dissect the molecular epidemiology and evolution of the main US epidemic CRKp sequence type (ST) 258. Interrogating strains and resistance harboring plasmids within our network of NYC hospitals we found that the majority of ST258 CRKp strains harbor one of three common plasmids carrying a particular class of carbapenemase enzyme, KPC. These data suggest the spread of CRKp is likely the consequence of plasmid-mediated gene transfer and subsequent clonal spread. We therefore hypothesize that this epidemic is primarily due to transmission of resistance harboring plasmids uniquely adapted to specific host genetic backgrounds. In Aim 1, we expand our NYC network to include a large US consortium and to examine the genomic epidemiology of CRKp strains and plasmids across the US. Aim 2 builds on the insights and techniques developed in our previous studies to interrogate the global epidemiology of CRKp via a large clinical isolate collection from over 62 countries, with the goal of constructing a phylogeographic map of strains, plasmids and carbapenemase genes. In this Aim we will also directly test the basis of CRKp strain-plasmid association by comparative transmission efficiency studies of different carbapenemase gene-harboring plasmids into diverse strain backgrounds. Using robust CRKp genomic data obtained in Aims 1 and 2, we will develop a rapid molecular detection assay to identify and track CRKp strains and plasmids in clinical settings. Aim 3 will characterize non-carbapenemase factors that contribute to high-level carbapenemase resistance in CRKp isolates, such as mutations in outer membrane proteins, which result in very poor clinical outcomes. Based on this characterization, some of these highly carbapenemase resistant strains will be selected to build a new, well-curated panel of strains for testing novel antibiotic agents. Taken together, ours is an innovative approach with the potential to make a substantial impact in the field of CRKp epidemiology, develop critically needed diagnostic platforms, and explore efficacy of novel antibiotics against these organisms.